Targeting the complexity of ERBB2 biology in gastroesophageal carcinoma.

ERBB2 is the most prominent therapeutic target in gastroesophageal adenocarcinoma (GEA). For two decades, trastuzumab was the only treatment available for GEA overexpressing ERBB2. Several drugs showing evidence of efficacy over or in complement to trastuzumab in breast cancer failed to show clinical benefit in GEA. This resistance to anti-ERBB2 therapy is peculiarly recurrent in GEA and is mostly due to tumor heterogeneity with the existence of low expressing ERBB2 tumor clones and loss of ERBB2 over time. The development of new ERBB2 testing strategies and the use of antibody-drug conjugates having a bystander effect are providing new tools to fight heterogeneity in ERBB2-positive GEA. Co-amplifications of tyrosine kinase receptors, alterations in mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) signaling pathways and in proteins controlling cell cycle are well known to contribute resistance to anti-ERBB2 therapy, and they can be targeted by dual therapy. Recently described, NF1 mutations are responsible for Ras phosphorylation and activation and can also be targeted by MEK/ERK inhibition along with anti-ERBB2 therapy. Multiple lines of evidence suggest that immune mechanisms involving antibody-dependent cell-mediated cytotoxicity are preponderant over intracellular signaling in anti-ERBB2 therapy action. A better comprehension of these mechanisms could leverage immune action of anti-ERBB2 therapy and elucidate efficacy of combinations associating immunotherapy and anti-ERBB2 therapy, as suggested by the recent intermediate positive results of the KEYNOTE-811 trial.

Pre-treatment tumor-infiltrating T cells influence response to neoadjuvant chemoradiotherapy in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a disease with dismal treatment outcomes. Response to neoadjuvant chemoradiation (CRT) varies greatly. Although the underlying mechanisms of CRT resistance are not identified, accumulating evidence indicates an important role for local antitumor immunity. To explore the immune microenvironment in relation to response to CRT we performed an in-depth analysis using multiplex immunohistochemistry, flow cytometry and mRNA expression analysis (NanoString) to generate a detailed map of the immunological landscape of pretreatment biopsies as well as peripheral blood mononuclear cells (PBMCs) of EAC patients. Response to CRT was assessed by Mandard's tumor regression grade (TRG), disease-free- and overall survival. Tumors with a complete pathological response (TRG 1) to neoadjuvant CRT had significantly higher tumor-infiltrating T cell levels compared to all other response groups (TRG 2-5). These T cells were also in closer proximity to tumor cells in complete responders compared to other response groups. Notably, immune profiles of near-complete responders (TRG 2) showed more resemblance to non-responders (TRG 3-5) than to complete responders. A high CD8:CD163 ratio in the tumor was associated with an improved disease-free survival. Gene expression analyses revealed that T cells in non-responders were Th2-skewed, while complete responders were enriched in cytotoxic immune cells. Finally, complete responders were enriched in circulating memory T cells. preexisting immune activation enhances the chance for a complete pathological response to neoadjuvant CRT. This information can potentially be used for future patient selection, but also fuels the development of immunomodulatory strategies to enhance CRT efficacy.

Characterizing diversity in the tumor-immune microenvironment of distinct subclasses of gastroesophageal adenocarcinomas.

BACKGROUND: Gastroesophageal adenocarcinomas (GEAs) are heterogeneous cancers where immune checkpoint inhibitors have robust efficacy in heavily inflamed microsatellite instability (MSI) or Epstein-Barr virus (EBV)-positive subtypes. Immune checkpoint inhibitor responses are markedly lower in diffuse/genome stable (GS) and chromosomal instable (CIN) GEAs. In contrast to EBV and MSI subtypes, the tumor microenvironment of CIN and GS GEAs have not been fully characterized to date, which limits our ability to improve immunotherapeutic strategies. PATIENTS AND METHODS: Here we aimed to identify tumor-immune cell association across GEA subclasses using data from The Cancer Genome Atlas (N = 453 GEAs) and archival GEA resection specimen (N = 71). The Cancer Genome Atlas RNAseq data were used for computational inferences of immune cell subsets, which were correlated to tumor characteristics within and between subtypes. Archival tissues were used for more spatial immune characterization spanning immunohistochemistry and mRNA expression analyses. RESULTS: Our results confirmed substantial heterogeneity in the tumor microenvironment between distinct subtypes. While MSI-high and EBV+ GEAs harbored most intense T cell infiltrates, the GS group showed enrichment of CD4+ T cells, macrophages and B cells and, in ∼50% of cases, evidence for tertiary lymphoid structures. In contrast, CIN cancers possessed CD8+ T cells predominantly at the invasive margin while tumor-associated macrophages showed tumor infiltrating capacity. Relatively T cell-rich 'hot' CIN GEAs were often from Western patients, while immunological 'cold' CIN GEAs showed enrichment of MYC and cell cycle pathways, including amplification of CCNE1. CONCLUSIONS: These results reveal the diversity of immune phenotypes of GEA. Half of GS gastric cancers have tertiary lymphoid structures and are therefore promising candidates for immunotherapy. The majority of CIN GEAs, however, exhibit T cell exclusion and infiltrating macrophages. Associations of immune-poor CIN GEAs with MYC activity and CCNE1 amplification may enable new studies to determine precise mechanisms of immune evasion, ultimately inspiring new therapeutic modalities.

Impact of genomic alterations on lapatinib treatment outcome and cell-free genomic landscape during HER2 therapy in HER2+ gastric cancer patients.

Background: To identify predictive markers for responders in lapatinib-treated patients and to demonstrate molecular changes during lapatinib treatment via cell-free genomics. Patients and methods: We prospectively evaluated the efficacy of combining lapatinib with capecitabine and oxaliplatin as first line neoadjuvant therapy in patients with previously untreated, HER2-overexpressing advanced gastric cancer. A parallel biomarker study was conducted by simultaneously performing immunohistochemistry and next-generation sequencing (NGS) with tumor and blood samples. Results: Complete response was confirmed in 7/32 patients (21.8%), 2 of whom received radical surgery with pathologic-confirmed complete response. Fifteen partial responses (46.8%) were observed, resulting in a 68.6% overall response rate. NGS of the 16 tumor specimens demonstrated that the most common co-occurring copy number alteration was CCNE1 amplification, which was present in 40% of HER2+ tumors. The relationship between CCNE1 amplification and lack of response to HER2-targeted therapy trended toward statistical significance (66.7% of non-responders versus 22.2% of responders harbored CCNE1 amplification; P = 0.08). Patients with high level ERBB2 amplification by NGS were more likely to respond to therapy, compared with patients with low level ERBB2 amplification (P = 0.02). Analysis of cfDNA showed that detectable ERBB2 copy number amplification in plasma was predictive to the response (100%, response rate) and changes in plasma-detected genomic alterations were associated with lapatinib sensitivity and/or resistance. The follow-up cfDNA genomics at disease progression demonstrated that there are emergences of other genomic aberrations such as MYC, EGFR, FGFR2 and MET amplifications. Conclusions: The present study showed that HER2+ GC patients respond differently according to concomitant genomic aberrations beyond ERBB2, high ERBB2 amplification by NGS or cfDNA can be a positive predictor for patient selection, and tumor genomic alterations change significantly during targeted agent therapy.

The tumor microenvironment in esophageal cancer.

Esophageal cancer is a deadly disease, ranking sixth among all cancers in mortality. Despite incremental advances in diagnostics and therapeutics, esophageal cancer still carries a poor prognosis, and thus, there remains a need to elucidate the molecular mechanisms underlying this disease. There is accumulating evidence that a comprehensive understanding of the molecular composition of esophageal cancer requires attention to not only tumor cells but also the tumor microenvironment (TME), which contains diverse cell populations, signaling factors and structural molecules that interact with tumor cells and support all stages of tumorigenesis. In esophageal cancer, environmental exposures can trigger chronic inflammation, which leads to constitutive activation of pro-inflammatory signaling pathways that promote survival and proliferation. Antitumor immunity is attenuated by cell populations such as myeloid-derived suppressor cells and regulatory T cells, as well as immune checkpoints like programmed death-1. Other immune cells such as tumor-associated macrophages can have other pro-tumorigenic functions, including the induction of angiogenesis and tumor cell invasion. Cancer-associated fibroblasts secrete growth factors and alter the extracellular matrix to create a tumor niche and enhance tumor cell migration and metastasis. Further study of how these TME components relate to the different stages of tumor progression in each esophageal cancer subtype will lead to development of novel and specific TME-targeting therapeutic strategies, which offer considerable potential especially in the setting of combination therapy.

Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated ß-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-ß-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

An integrative analysis reveals functional targets of GATA6 transcriptional regulation in gastric cancer.

Lineage-restricted transcription factors (TFs) are frequently mutated or overexpressed in cancer and contribute toward malignant behaviors; however, the molecular bases of their oncogenic properties are largely unknown. As TF activities are difficult to inhibit directly with small molecules, the genes and pathways they regulate might represent more tractable targets for drug therapy. We studied GATA6, a TF gene that is frequently amplified or overexpressed in gastric, esophageal and pancreatic adenocarcinomas. GATA6-overexpressing gastric cancer cell lines cluster in gene expression space, separate from non-overexpressing lines. This expression clustering signifies a shared pathogenic group of genes that GATA6 may regulate through direct cis-element binding. We used chromatin immunoprecipitation and sequencing (ChIP-seq) to identify GATA6-bound genes and considered TF occupancy in relation to genes that respond to GATA6 depletion in cell lines and track with GATA6 mRNA (synexpression groups) in primary gastric cancers. Among other cellular functions, GATA6-occupied genes control apoptosis and govern the M-phase of the cell cycle. Depletion of GATA6 reduced the levels of the latter transcripts and arrested cells in G2 and M phases of the cell cycle. Synexpression in human tumor samples identified likely direct transcriptional targets substantially better than consideration only of transcripts that respond to GATA6 loss in cultured cells. Candidate target genes responded to the loss of GATA6 or its homolog GATA4 and even more to the depletion of both proteins. Many GATA6-dependent genes lacked nearby binding sites but several strongly dependent, synexpressed and GATA6-bound genes encode TFs such as MYC, HES1, RARB and CDX2. Thus, many downstream effects occur indirectly through other TFs and GATA6 activity in gastric cancer is partially redundant with GATA4. This integrative analysis of locus occupancy, gene dependency and synexpression provides a functional signature of GATA6-overexpressing gastric cancers, revealing both limits and new therapeutic directions for a challenging and frequently fatal disease.

Research use of linked health data--a best practice protocol.

OBJECTIVE: This article outlines a protocol for facilitating access to administrative data for the purpose of health services research, when these data are sourced from multiple organisations. This approach is designed to promote confidence in the community and among data custodians that there are benefits of linked health information being used and that individual privacy is being rigorously protected. BACKGROUND: Linked health administration data can provide an unparalleled resource for the monitoring and evaluation of health care services. However, for a number of reasons, these data have not been readily available to researchers. In Australia, an additional barrier to research is the result of health data sets being collected by different levels of government - thus all are not available to any one authority. To improve this situation, a practical blue-print for the conduct of data linkage is proposed. This should provide an approach suitable for most projects that draw large volumes of information from multiple sources, especially when this includes organisations in different jurisdictions. CONCLUSIONS AND IMPLICATIONS: Health data, although widely and diligently collected, continue to be under-utilised for research and evaluation in most countries. This protocol aims to make these data more easily available to researchers by providing a controlled and secure mechanism that guarantees privacy protection.

Changing treatments for primary urolithiasis: impact on services and renal preservation in 16,679 patients in Western Australia.

OBJECTIVES: To examine changing treatments for the primary presentation of urinary lithiasis and their effects on re-admissions, repeat procedures, cumulative hospital use and renal preservation. PATIENTS AND METHODS: Linked hospital morbidity records were used to identify first-time admissions for renal and ureteric calculi from 1980 to 1997 in the population of Western Australia. The cases were followed to mid-1999 and actuarial methods used to estimate risks of further hospital admissions and procedures, including the loss of a renal moiety. RESULTS: Between 1980 and 1997 the total rate of inpatient procedures for urinary stones more than doubled, at a time when the rate of first-time hospital admissions increased by only 13% and the conservative management of stones remained constant at approximately 59%. The predominant procedure for stone management was initially open lithotomy, replaced in the early 1980s by percutaneous nephrolithotomy and soon supplemented by extracorporeal shock wave lithotripsy (ESWL). The changes in technology led to a four-fold increase in procedural re-admissions within 30 days of primary separation. This was a result of repeated, staged or postponed interventions, often involving the use of stents or a second treatment with ESWL. The risk of surgical intervention decreased from 48% to 32%, whilst the cumulative length of stay over the first year decreased from 7.8 to 3.9 days. The risk of kidney loss declined significantly from 2% to <0.1% during the period. CONCLUSIONS: The main reason for more interventions were short-term procedural re-admissions. ESWL reduced the need for invasive procedures and decreased cumulative hospital stay, despite more re-admissions. Renal preservation improved by a factor of 10.

Population-based outcomes after 28,246 in-hospital vasectomies and 1,902 vasovasostomies in Western Australia.

OBJECTIVES: To examine trends in vasectomy and vasovasostomy, and the surgical complications and factors associated with reversal after vasectomy, and paternity after vasovasostomy. PATIENTS AND METHODS: Procedure rates were estimated from 1980 to 1996 in the population of Western Australia. Linked hospital morbidity records were used in the follow-up of men after vasectomy to estimate the risks of complications and reversals. Records of vasovasostomies were linked to the paternity field on birth registrations. Independent effects of the study factors were examined using Cox regression. RESULTS: There was little net change in vasectomy rates, whereas vasovasostomy rates increased in men aged 30-49 years. Risks of surgical complications were low and decreased for vasovasostomy. At 12-15 years after vasectomy, the risk of reversal levelled at 2. 4% in the total cohort and at 11.1% in men aged 20-24 years. The risk of vasovasostomy was 69% greater after vasectomy performed in 1994-96 than in 1980-84 (P = 0.011). The factors strongly associated with reversal were age < 30 years and being single, divorced or separated at the time of vasectomy. Paternity was achieved after an estimated 53% of vasovasostomies. Successful reversal was more likely if the man was younger at vasectomy and the time elapsed was comparatively short. Compared with vasovasostomies performed in 1980-84, the success rate of those in 1994-96 was almost four times higher. CONCLUSION: Population rates of vasectomy are stable but the risk of seeking a reversal has increased. Outcomes after vasovasostomy have improved. Care should be taken during the counselling of men before vasectomy, and especially in those aged <30 years.

Population-based linkage of health records in Western Australia: development of a health services research linked database.

OBJECTIVES: To introduce the Western Australian Health Services Research Linked Database as infrastructure to support aetiologic, utilisation and outcomes research. To compare the study population, data resources, technical systems and organisational supports with international best practice in record linkage and health research. METHOD AND RESULTS: The WA Linked Database systematically links the available administrative health data within an Australian State of 1.7 million people. It brings together, initially, six core data elements (birth records, midwives' notifications, cancer registrations, in-patient hospital morbidity, in-patient and public out-patient mental health services data and death records). It will be updated regularly and is designed, in future extensions, to include data on primary, residential and domiciliary care and health surveys. Linkage uses probabilistic matching of patient names and other identifiers. Geocodes for spatial analysis are assigned using address linkage and mapping software. By June 1997, the project had taken 2 1/2 years to develop the system and link seven million core data records from 1980 to 1995. CONCLUSIONS: The system is consistent with international benchmarks, from four centres of excellence, for the study population, core datasets, matching and geocoding, and collaborative networks. There are prospects to redress deficiencies in primary medical contact and other data resources, validation studies, tracing systems and a more supportive legal framework. IMPLICATIONS: The WA Linked Database will be used in combination with medical record audits to provide a comprehensive evaluation of health system performance.

Validation of linked administrative data on end-stage renal failure: application of record linkage to a 'clinical base population'.

OBJECTIVE: To evaluate the use of record linkage to monitor the occurrence of end-stage renal failure in Western Australia in 1980-94. METHODS: A clinical base population of 1,046 patients was identified from the Western Australian (WA) Health Services Research Linked Database. To exclude acute renal failure, patients were selected if they received in-hospital renal dialysis on more than 10 occasions over more than 28 days in 1980-94. Estimates of annual incident and prevalent cases were validated against the ANZDATA dialysis and transplant register. Reasons for discrepancy were investigated by an ad hoc linkage between the two data sources. RESULTS: The WA Linked Database counted slightly fewer incident cases (-7%) and slightly more prevalent cases (+7%) than the ANZDATA Register. The Linked Database identified 97% of cases on the ANZDATA Register, but this fell to 83% post case definition, probably due to patients receiving home-based dialysis failing to meet our case definition. ANZDATA correctly identified 90% of cases in the linked file. CONCLUSION: Trends in end-stage renal failure from 1986 to 1994, based on the Linked Database, were the same as those reported from purpose-designed disease registers. IMPLICATIONS: Linked administrative data provide a valid and efficient means to plan and evaluate many of the routine aspects of renal dialysis and transplant services.

Trends in repeat prostatectomy after surgery for benign prostate disease: application of record linkage to healthcare outcomes.

OBJECTIVE: To compare the risk of repeat prostatectomy for benign prostatic hyperplasia (BPH) in a population-based cohort of 19 598 men in Western Australia treated by transurethral resection of the prostate (TURP) or open prostatectomy over a 16-year period. PATIENTS AND METHODS: The Western Australian Health Services Research Linked Database was used to extract all hospital morbidity data, death records and prostate cancer registrations for men who had prostate surgery for BPH in 1980-95. The cumulative incidence of first repeat prostatectomy calculated using the actuarial life-table and incidence-rate ratios of the first repeat prostatectomy, comparing TURP and open prostatectomy, were obtained using Cox regression. RESULTS: The cases comprised 18 464 TURPs and 1134 open prostatectomies, from which there were 1095 subsequent repeat prostatectomies. After adjustment for calendar time, age and admission type, the incidence rate of the first repeat prostatectomy was up to 2.30 times higher (95% confidence interval, 1.62-3.27) after initial TURP than for initial open prostatectomy. The absolute risks at 8 years for TURP was 6.6%, and was 3.3% for open prostatectomy. CONCLUSION: The absolute risk of a repeat prostatectomy for TURP and open prostatectomy were consistent with the best reported international experience. There was evidence that the risk in 1990-95 had declined compared with earlier periods, despite a shift towards more closed procedures. The differential risks of repeat prostatectomy should be explained to patients and considered in the development of clinical guidelines, notwithstanding the advantages of TURP over open prostatectomy in terms of surgical morbidity and cost.

Mortality and prostate cancer risk in 19,598 men after surgery for benign prostatic hyperplasia.

OBJECTIVES: To examine postoperative mortality and prostate cancer risk after the first prostatectomy for benign prostatic hypertrophy over a 17-year period in a population-based cohort of men in Western Australia, using improved methods to adjust for comorbidity. PATIENTS AND METHODS: The relative survival from death and prostate cancer incidence was calculated against the background population rates. The outcomes of transurethral resection of the prostate (TURP) and open prostatectomy (OP) were compared adjusting for calendar year, age, admission type and comorbidity using Cox regression. Fractional polynomials were used to take account of nonlinearity in confounder effects. RESULTS: At 10 years, the relative survival was 116.5% in TURP patients and 123.5% after OP. Adjusting only for confounding by age, calendar year and admission type, TURP had a higher mortality rate than OP (rate ratio, RR, 1. 20; 95% confidence interval 1.08-1.34). The RR fell to 1.10 (0.99-1. 23) after adjustment for comorbidity and to 1.07 (0.95-1.19) when accounting for nonlinearity. The relative survival from the incidence of prostate cancer at 10 years was 103.7% after TURP and 104.5% after OP. The RR adjusted for age and calendar year was 1.44 (0.94-2.21) for incidence and 1.37 (0.81-2.29) for prostate cancer mortality. CONCLUSION: There is at most a small and clinically unimportant excess mortality risk from TURP; any difference could be due to a protective effect of OP on the long-term risk of prostate cancer and a lower rate of repeat prostatectomy.

Population-based record linkage study of the incidence of abdominal aortic aneurysm in Western Australia in 1985-1994.

BACKGROUND: The incidence of abdominal aortic aneurysm (AAA) has increased steadily during the past 30 years. METHODS: Trends in the incidence and surgical intervention for AAA in Western Australia were reviewed for the interval 1985-1994. A population-based health database was used to link morbidity and mortality records of all patients aged 55 years or more who died from rupture or were admitted and treated surgically for AAA. Three groups were separated for analysis: patients with a ruptured AAA, those admitted for elective repair and those admitted as an emergency with an acute (non-ruptured) aneurysm. RESULTS: There was a decline in the incidence of both emergency and elective procedures for AAA after 1992. While the mortality rate from ruptured AAA has also fallen since 1991, the overall case fatality rate for ruptured AAA has fallen by only 1.3 per cent (from 80.7 to 79.3 per cent). CONCLUSION: The decline in mortality rate and emergency procedures may result from a fall in the incidence of ruptured AAA, due to an increasing rate of elective surgery before 1992. The decline in elective procedures from 1992 may be due to a fall in the prevalence of AAA owing to high rates of elective surgery, or to a fall in the incidence of the disease itself.

Hospitalisation of the elderly during the last year of life: an application of record linkage in Western Australia 1985-1994.

STUDY OBJECTIVE: To measure the trend, pattern, and cost of time spent in hospital during the last year of life in Western Australia and to identify trends in the place of death. The results were compared with those reported from the Oxford Record Linkage Study. DESIGN: Mortality records for those aged 65 years and over were linked to inpatient hospital morbidity records with a date of separation within one year before death. Comparative inpatient resource utilisation was estimated using ANDRG 3.0 cost weights for Australian public hospitals. SETTING: Western Australia. PARTICIPANTS: All 68,875 persons aged 65 years and over who died between 1 January 1985 and 31 December 1994. MAIN RESULTS: Increasing proportions of all age groups (65-74, 75-84, and 85+ years) were admitted to hospital at least once in the year before death during 1985-94, but the chance of admission decreased with age. There was a trend towards a greater number of shorter admissions per person. Total bed days per person showed no significant increase, except at ages 65-74 years. Total inpatient resource utilisation during the last year of life was lowest and remained constant in those aged 85 years and over, while increasing gradually (3.7% per annum) in the younger elderly. The Western Australian population spent more time in hospital in the last year of life at ages 65-74 years, but the advanced elderly spent less time in hospital, when compared with the Oxford Region. CONCLUSIONS: Recent gains in life expectancy and higher per capita health expenditure have not been accompanied by more time spent in hospital during the last year of life at ages 75+ years. International differences between Western Australia and Oxford can be explained by differences in aged care provision.